59º Congresso Brasileiro de Genética
Resumos do 59o Congresso Brasileiro de Genética • 16 a 19 de setembro de 2013
Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
110
The pathogenesis-related protein PR-4 from
Theobroma cacao is involved in the defense
responses of cacao against Moniliophthora
perniciosa
Silva, EMA1; Menezes, SP1; Lima, EM1; Sousa, AO1; Gramacho, KP2; Gesteira, AS3; Micheli, F1,4.
Universidade Estadual de Santa Cruz, Centro de Biotecnologia e Genética, Rodovia Ilhéus-Itabuna, Km 16, 45662-000,
Ilhéus-BA, Brasil; 2CEPLAC/CEPEC, 45600-970, Itabuna-BA, Brasil; 3Embrapa Mandioca e Fruticultura, Rua Embrapa, s/nº,
44380-000, Cruz das Almas-BA, Brasil; 4CIRAD, UMAR AGAP, Avenue Agropolis, Montpellier, France.
1
Keywords: gene expression, antifungal activity, witches’ broom disease, Theobroma cacao, TcPR-4
The pathogenesis-related proteins class 4 (PR-4) is known to be involved in plant defense response. The objective
of this study was to evaluate the temporal expression of the TcPR-4 gene after inoculation with Moniliophthora
perniciosa. Plantlets of Theobroma cacao L. varieties Catongo (susceptible to M. perniciosa) and TSH1188 (resistant
to M. perniciosa) were inoculated by the droplet method with a basidiospore suspension (2.105 basidiósporo.ml-1) of
M. perniciosa. After inoculation, the plantlets were kept for 24h at 25±2ºC and 100% humidity. Apical meristems
were harvested in triplicates at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and 90 days after
inoculation (dai). Non-inoculated plants (controls) were kept and harvested in the same conditions at 24 hai, 72 hai,
30 dai, 60 dai and 90 dai. Total RNA was extracted from macerated samples using the RNAqueous Kit® (Ambion). The
synthesis of the first cDNA strand was done using the Revertaid Fisrt Strand cDNA Synthesis Kit (Thermo Scientific,
Fermentas). Real-time quantitative analysis of TcPR-4b expression was performed using standard settings of the ABI
PRISM 7500 and Sequence Detection System (SDS) software, version 1.6.3 (Applied Biosystems). The expression
level of Tc-PR4b was analyzed on triplicates with the comparative Ct method (2-ΔΔCt) using malate dehydrogenase and
actin as endogenous reference genes, and non-inoculated plants (control) were used as a calibrator. In the early stages of
infection, an increase of TcPR-4b expression at was observed 48 hai in both varieties TSH1188 and Catongo. After the
initial stage, the expression of TcPR-4b was observed in all times in the resistant variety, while in the susceptible one the
TcPR-4b expression was concentrated in the final stages of infection. In parallel, The recombinant protein TcPR-4b,
overexpressed in the pET28a plasmid,was used in antifungal tests against M. perniciosa (pseudo-colony method). The
recombinant protein used at different concentrations shows antifungal activity against M. perniciosa. The data generated
in this study may help to elucidate mechanisms of the TcPR-4b action during the interaction cacao-M. perniciosa.
Financial Support: CNPq, BNB, FINEP/RENORBIO, CAPES, Cirad.